Three distinct classes of single-cell clones were derived from the B16 mouse melanoma: (1)\"null" clones produce tumors in syngeneic C57BL/6 mice but form few or no lung colonies following injection into the tail vein and fail to metastasize; (2)\colonizing clones form numerous lung colonies following intravenous injection but do not metastasize significantly; and (3)\metastatic clones spontaneously metastasize to the lungs but are inefficient at forming lung colonies when injected in the tail vein. Utilizing representative clones, the basis for distinction between tumorigenicity, lung colonization, and lung metastasis will be probed to provide novel information about the occurrence and mechanism of spontaneous metastasis. The capacity of tumor cells growing subcutaneously to circulate within blood vessels, monitored by vascular perfusion, was demonstrated for metastatic tumors but not for tumors initiated by colonizing clones. The ability of cells from subcutaneous tumors to metastasize to various organs was assessed following surgical excision of tumors or by bioassay of organs. The pattern of metastasis was as widespread and indiscriminate as human malignant melanoma, involving lungs, lymph nodes, brain, kidneys, adrenals, ovaries, liver, and intestines. Metastasis results from nonspecific trapping in the first capillary bed encountered, first in the lungs and then from established lung metastases to systemic organs or organ implants. No specific lung tropism was demonstrable to ectopic organ implants or in parabiosed mice, and metastases failed to exhibit organ-specific tropism. Metastatic activity has been generated from null clones in vitro and in vivo and is related to a tumor growth rate intermediate between null and colonizer tumors; metastatic cells are not the most rapidly and effectively growing tumor cells. The ultimate goal of this research is to provide information to permit the designing of successful immunotherapy of metastatic tumors. (S)